The long-term goal of this project is to understand the molecular mechanism that governs the reactivation of Herpes Simplex Virus type 1 (HSV-1) from latency. Work during the previous funding period has focused on the role of the reactivation critical region (rcr) in this process, and whether latent gene expression was regulated in an epigenetic manner. Our key findings were that: 1) the suppression of HSV-1 lytic genes during latency is associated with histone modifications and not DNA methylation;2) the histones associated with the rcr are maintained in a hyperacetylated state during latency;3) CTCF-containing chromatin insulator-like elements flank the region of hyperacetylation, preventing the spread of transcriptionally active chromatin to the surrounding lytic genes and 4) following reactivation stimulus rapid deacetylation of the LAT enhancer is followed by a dramatic decrease in LAT abundance and acetylation of the ICP0 promoter. These data suggest that the rcr functions as a regulatory element that controls LAT and ICP0 transcription at the level of chromatin. Specifically, the LAT enhancer portion of the rcr and a chromatin insulator act together as a switch that regulates LAT and ICP0 in a bi-phasic manner. Based on this new data and drawing from chromatin-level regulatory models employed by cellular chromosomes, we have refined our model for the LAT region's role in reactivation. This model views the LAT region as a regulatory locus that controls the transcriptional permissiveness of ICP0 through epigenetic regulation of histone modifications. This regulatory locus consists of at least three components: 1) the LAT enhancer that controls histone modifications, and has the potential to regulate transcription of both the LAT and ICP0;2) the CTCF-insulator elements which flank the rcr and regulate the extent of the LAT enhancer's influence, and 3) transcription of the LAT RNA itself, which we hypothesize regulates the insulator. This project will seek to prove our overall hypothesis that regulation of these three components governs the ability of HSV-1 to reactivate. HSV-1 is a persistent infection of humans that causes significant morbidity and mortality. Understanding the mechanism of how stress induces reactivation could lead to new therapies to treat and prevent recurrent disease. At a broader level, understanding the mechanism by which HSV regulates transcription via modulation of chromatin structure will increase our understanding of similar processes and pathways in cellular chromosomes, potentially leading to new therapies for developmental disorders and cancer.